Project Summary/Abstract Our long-term goal is to understand how interactions between elements in noncoding regions of vertebrate mRNAs and their cognate binding proteins integrate signals from disparate stimuli to control translation. Tran- script-selective translationl control is mediated by interactions of RNA-binding proteins to sequence/structural elements in the 5'- or 3'-untranslated region (UTR) of target transcripts. Recently, an additional layer of com plexity has been recognized in which element pairs act as condition-dependent RNA switches. For example, riboswitches are proximate structural elements in the UTR of multiple bacterial mRNAs that undergo conforma- tional changes in response to specific metabolites. We have reported an analogous, stimulus-dependent switch in the 3'UTR of human vascular endothelial growth factor (VEGF)-A mRNA. VEGF-A mRNA contains adjoining elements that function as a novel stimulus-dependent, protein-directed RNA switch that exists in two metastable conformations: a translation-silencing and a translation-permissive conformer. The binary switch is controlled by integration of two signals, interferon (IFN)- and hypoxia, that regulate the amount or activity of the binding factors. Upon cell stimulation by IFN-, phosphorylation of Glu-Pro tRNA synthetase (EPRS) initiates formation of the GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex. EPRS binds a defined, GAIT element in the VEGF-A mRNA 3'UTR, stabilizing the translation-silencing conformer and inhibiting translation. However, superimposition of hypoxia on IFN- stimulation induces phosphorylation of hnRNP L at Tyr359 that initiates assembly of a newly discovered 3-component HILDA complex that binds a CA-rich element directly upstream of the GAIT element, stabilizing the translation-permissive conformer and allowing VEGF-A expression. We propose the following specific hypothesis: Myeloid cells integrate signals from IFN- and hypoxia by inducing Tyr359 phosphorylation of hnRNP L and assembly of the HILDA complex, which in turn directs an RNA switch in the 3'-UTR of VEGF-A and other inflammation-related mRNAs to regulate translation. We will test this hypothesis by pursuing the following Specific Aims: Aim 1: Investigate molecular mechanisms regulating hnRNP L expression and localization; Aim 2: Determine the functions of HILDA components in regulating the RNA switch; Aim 3: Identify novel transcripts controlled by protein-directed RNA switches. We suggest that the switch evolved to maintain VEGF-A expression and angiogenesis in hypoxic, inflammatory tissues. Tumors, also residing in hypoxic, inflammatory sites, may take advantage of the VEGF-A switch to stimulate inward blood vessel growth to provide nourishment and permit tumor growth. Thus, the VEGF-A switch represents a novel therapeutic target to specifically inhibit tumor macrophage expression of VEGF-A. We also speculate that the VEGF-A switch may represent the founding member of a family of protein-directed RNA switches in vertebrates that integrate physiological or pathological stimuli to control gene expression.